Development efforts have been made in many different fields of industry for biosensors incorporating an enzyme which makes a specific reaction with a specific substrate. A representative example of such biosensors is a glucose sensor utilized mainly in the field of medical care.
The glucose sensor establishes a reaction system which includes an enzyme and an electron transfer material. When using the glucose sensor, an amperometric method for example is employed to quantitate glucose. The enzyme is provided by e.g. glucose oxidase (GOD) and glucose dehydrogenase (GDH).
GOD has high substrate specificity to glucose, high thermal stability, and is less expensive than other enzymes because it can be mass-produced industrially. A shortcoming on the other hand is that reaction systems involving GOD are highly sensitive to oxygen dissolved in the sample, and so the dissolved oxygen can affect the measurement.
On the other hand, reaction systems involving GDH are not susceptible to dissolved oxygen in the sample. For this reason, reaction systems which utilize GDH allow accurate measurement of the glucose level even if the measurement is made in an environment poor in oxygen partial pressure, or if the measurement is made to a high-concentration sample which has a high oxygen demand. Shortcomings of GDH include poor thermal stability and lower substrate specificity than GOD.
Under these circumstances, efforts have been made in search for an enzyme which covers the shortcomings of both GOD and GDH. As disclosed in the International Disclosure WO02/36779, Hayade separated a new strain (Burkholderia cepacia KS1) from soil near a hot spring, and obtained a new GDH from the strain. This GDH included α, β, γ subunits (hereinafter called “CyGDH”), had a high rate of reaction with electron transfer materials, and sufficient thermal stability, and so was suitable for use in glucose sensors.
When utilizing CyGDH in glucose sensors, CyGDH must be purified from an enzyme solution which contains CyGDH. Enzyme is usually purified by means of liquid chromatography, so the inventor et al. of the present invention followed a common method of hydrophobic chromatography and anion exchange chromatography in an attempt to process the enzyme solution. However, it was not possible to purify CyGDH to a high level of purification, as SDS-PAGE examinations revealed that the obtained enzyme solution contained a number of different proteins in addition to α, β, γ subunits.